PROJECT SUMMARY AND ABSTRACT High grade gliomas including recurrent glioblastoma in adults and brain stem gliomas in children are invariably fatal. Harnessing the immune system using immunotherapy has made significant strides towards treatment against solid tumors in both murine and human systems, however, in the setting of malignant gliomas, the majority of hosts still succumb to disease. Two major contributors to treatment resistance in immunotherapy against malignant gliomas are 1) tumor heterogeneity, and 2) immunosuppression within both the tumor microenvironment and the hosts' hematopoietic system. Our group has recently demonstrated that concomitant transfer of hematopoietic stem cells (HSC) with immunotherapy leads to overcoming of treatment failure to both adoptive cellular therapy and checkpoint inhibition by directly addressing these two challenges (Flores et al. Nature Communications. In press). A novel and innovative way to address tumor heterogeneity is by leveraging HSC co-transfer with adoptive cellular therapy. Transferred HSCs migrate to intracranial tumor within hours and differentiate into dendritic cells that capture tumor antigens in situ and present to both adoptively transferred and host tumor infiltrating lymphocytes, demonstrably perpetuating T cell activation within the otherwise immunosuppressive tumor microenvironment. In addition, our recently published data demonstrates a novel approach of concomitant transfer of HSCs with immunotherapy shuts down multiple key immunoregulatory pathways within the tumor microenvironment simultaneously including PD-L-1, TGF?, iNOS, and IDO-1. HSCs also supplant endogenous host MDSCs and TAMs within malignant glioma decreasing overall regulatory cells. We suspect that the HSC transfer impacts either the proliferation or recruitment of endogenous suppressive cells to tumor. This reduction in suppressive pathways is likely a major contributor to the maintenance of T cell activation. This proposal will focus on the mechanisms by which HSCs supplant endogenous immunoregulatory cells within the tumor and increase activation of adoptively transferred tumor-reactive T cells within the tumor microenvironment. As HSCs turn off immunosuppressive regulatory pathways, they concurrently increase tumor- reactive T cell activation. If successful, ongoing and near future clinical studies at our institution can increase efficacy of adoptive cellular therapy in children with malignant gliomas. Our HYPOTHESIS is that HSCs overcome treatment resistance to adoptive cellular therapy by continual cross-priming of T lymphocytes while shutting down regulatory pathways both within the tumor microenvironment. The AIMS of this project are to:AIM 1. Determine if HSC-derived dendritic cells have the capacity to present antigens from escaped tumor after adoptive cellular therapy; AIM 2. Evaluate the mechanisms by which HSCs simultaneously target multiple modulatory pathways within the tumor microenvironment; AIM 3. Understand resistance mechanisms to HSC + adoptive cellular therapy in subjects with non-curative treatment.